Summary of work: This project focusses on the signal transduction pathways mediating stress responses in mammalian cells. Studies over the past year have concentrated on three topics. (1) Identification of upstream regulators of ERK signaling in response to arsenite. We have previously demonstrated that arsenite can differentially activate ERK, JNK and p38 MAP kinases. The activation of ERK is dependent on Ras. Now we have found that arsenite can stimulate the tyrosine phosphorylation of Shc and its interaction with the adaptor protein Grb2. Tyrosine phosphorylation of Shc was concommitant with tyrosine phosphorylation of an unidentified high molecular weight protein, with which it interacts. This protein may function as an activator of Shc during stress, initiating the Ras/ERK signaling cascade. (2) p90RSK activation in response to stress and its relationship with IkB. p90RSK is a serine/threonine protein kinase which lies downstream of ERK, and is therefore activated by mitogenic stimuli. We found that p90RSK can be activated by many stressful conditions including arsenite, H2O2 and methyl methanesulfonate. p90RSK activation was found to correlate with the phosphorylation and degradation of IkB, an inhibitor of the transcription factor NF-kB. p90RSK may function as an IkB kinase and contribute to NF-kB activation during stress. (3) Age-associated decline in p70S6k in EGF-stimulated hepatocytes. Aging is associated with a decline in the proliferative capacity of cells. Previously we showed that this was correlated with decreased ERK activation by mitogenic stimuli. We have now observed that there is also a significant decline in the activity of p70S6k with aging. Thus, defects in at least two distinct signal transduction pathways may account for the age-associated defect in proliferative capacity. Analysis of the upstream regulators common to both pathways may provide important insights into the basic mechanisms of the aging.